ES2399667T3 - New agents for the treatment of disorders connected with impaired neurotransmission - Google Patents

Abstract

Use of an orally administrable dietary or pharmaceutical composition comprising an extract obtained from plants of the genus Origanum by extraction with a solvent or supercritical fluids, hydro-distillation to obtain essential oils, or extraction / distillation with hot gases in a process for: maintenance or the improvement of attention and concentration, memory and ability to remember, learning ability, language processing, problem solving and intellectual functioning, improvement of short-term memory, increased mental alertness, increased ability to concentrate and mental acuity, intensification of mental vigilance, reduction of mental fatigue, reinforcement of mental health, maintenance of a balanced cognitive function, regulation of hunger and satiety and regulation of motor activity, use as an antidepressant , mood / vitality enhancer, stress reliever, d reducer e anxiety, stress reducer, sadness reducer, unhappiness / discontent reducer, irritability reducer, ladisforia reducer, obsessive-compulsive behavior reducer, and / or relaxing, or use as a preventative or normalizer of alterations of circadian rhythms, with the proviso that the composition does not include the combination of oregano and Crataegus extracts.

Description

New agents for the treatment of disorders connected with impaired neurotransmission

The present invention relates to the use of oregano extracts and / or volatile components of oregano extract as agents for use in a method of treating disorders connected with impaired neurotransmission, which is reduced, as detailed herein.

It has been found, in accordance with this invention, that such extracts are inhibitors of serotonin reabsorption, inhibitors of norepinephrine reabsorption, and inhibitors of dopamine reabsorption. Thus, they can mimic the action of pharmaceutical products that are dual absorption inhibitors and triple absorption inhibitors. Therefore, these extracts and their volatile materials are particularly effective in the

10 treatment of mood disorders, including depression and anxiety, and can be used to regulate biorhythms in humans. They also have numerous veterinary uses, and can be used to prevent or treat the behavioral or physiological consequences of stress.

WO 95/05838 discloses the use of a volatile vegetable oil derivable from clove, nutmeg, pepper, thyme, paprika, oregano, marjoram, basil and French tarragon or a constituent thereof (e.g. linalol, tujona,

15 canphene, carvacrol, and thyme oil thymol) to combat deleterious changes in the peripheral nervous system (such as morphology, structure and amount of tissue). There is no doctrine about the use of the use of these substances affecting the biochemical processes in the brain.

WO 01/45780 discloses the use of phototherapy, optionally in combination with aromatherapy, where oregano is one of the possible ingredients, for treating sleep disorders combined with nervousness. So

20 thus, the composition is administered by olfactory means.

Field of the present invention

This invention relates to the use of oregano extracts and / or their volatile components for the manufacture of compositions for the treatment and prevention of disorders connected with released or reduced neurotransmission. Thus, in one aspect the present invention relates to dietary or pharmaceutical compositions or

25 veterinarians, which comprise at least one extract of oregano or one of its volatile components for use in a process for the maintenance or improvement of attention and concentration, memory and ability to remember, learning ability, language processing , problem solving and intellectual functioning, improvement of short-term memory,

30 increased mental alertness, increased concentration and mental acuity, intensified mental vigilance, reduced mental fatigue, reinforced mental health,

35 maintaining balanced cognitive function, regulating hunger and satiety and regulating motor activity, use as an antidepressant, mood / vitality enhancer, stress reliever, anxiety reducer, stress reducer, sadness reducer , unhappiness / discontent reducer, irritability reducer, dysphoria reducer, obsessive-compulsive behavior reducer, and / or relaxing, or

40 use as a preventive or normalizer of the alterations of circadian rhythms, with the proviso that the composition does not include the combination of oregano and Crataegus extracts.

Surprisingly, it has been found that oregano extracts and their volatile components of this invention act as serotonin reuptake inhibitors, thereby prolonging the time that serotonin is available for neurotransmission. This leads to a balance of mood and stress relief effect. Additionally, oregano extracts and their volatile components of this invention are also effective as norepinephrine reuptake inhibitors, dopamine resorption inhibitors, and / or serotonin, noradrenaline and / or dopamine dual resorption inhibitors, or triple inhibitors of the reabsorption of norepinephrine, serotonin and dopamine. Thus, they are particularly useful for the treatment of depression, anxiety, or a combination thereof, mediated by the neurotransmission of serotonin or

50 norepinephrine or a combination thereof.

The main neurotransmitters are serotonin, dopamine, norepinephrine, acetylcholine, glutamate, and gammaaminobutyric acid. The neurotransmitters of particular relevance for mood-related disorders are serotonin, norepinephrine and dopamine. The increase in neurotransmission is achieved by increasing the concentration of the neurotransmitter in the synaptic cleft, thus making it available for neurotransmission.

55 increased or prolonged by inhibition of reabsorption at the end of the pre-synaptic nerve, or by prevention of catabolism of neurotransmitters by inhibition of degrading enzymes such as monoaminooxidases A and B.

DEFINITIONS

The terms "impaired neurotransmission" and "reduced neurotransmission" are used interchangeably throughout the present application. They are used in the present application in accordance with their meaning well known to those skilled in the art, and refer to a state of dysregulation of neurotransmission,

5 that can occur at the level of biosynthesis, processing, storage, release, resorption and fixation of neurotransmitter receptors. Impaired neurotransmission, in particular a reduction in neurotransmission, can manifest itself in animals including humans as a disturbance of behavior, emotions, mood and thought processes, for example, in one of several types of depression.

10 The term "oregano extract and / or its volatile components" is intended to comprise not only complete mixtures of extractable compounds but also only volatile plant compounds considered in isolation or in any combination between them. The most important volatile components of oregano extracts according to the present invention are: carvacrol, thymol, thimoquinone and thimoquinol. Examples of additional volatile components of oregano extracts are:

15 4-tert-butylphenol; 2,3-diisopropyl-5-methylphenol; 2,4-diisopropyl-3-methylphenol; 2,4-diisopropyl-5-methylphenol; 2,5-diisopropyl-3-methylphenol; 2,5-diisopropyl-4-methylphenol; 2,6-diisopropyl-3-methylphenol and p-ment-3-in-1-ol.

The term "oregano extracts" of the present invention does not encompass hot aqueous teas or extracts produced from fresh or dried leaves or any other parts of oregano species, as well as teas containing only trace amounts of the volatile components.

The extracts obtained by steam distillation are, however, within the scope of the present invention. Such extracts generally contain volatile compounds that do not readily degrade. Distilled oils contain scarcely thymoquinone and other volatile materials, since they degrade more rapidly during steam distillation. However, they may contain high amounts of carvacrol. Extracts with SFCO2 are especially preferred for their stability (up to 5 years in closed containers).

25 The term "animals" includes humans, and encompasses mammals, fish and birds. Preferred are: humans, pets or pets, farm animals, and animals used in the fur industry.

The term "farm animals" includes: fish, such as salmon and trout, farmed animals such as shrimp, pigs, horses, ruminants (cattle, sheep, goats) and poultry (such as geese, chickens, chicks roast, laying hens, quail, ducks, and turkeys). Poultry, cattle, cattle are preferred,

30 sheep, goats and pigs.

"Pets" or "pets" include dogs, cats, birds, aquarium fish, guinea pigs, rabbits (jack), hares and ferrets. Dogs and cats are preferred.

"Animals used in the fur industry" include minks, foxes, and hares.

The term "dietary compositions" includes any type of nutritional product, such as food / feedstuffs and 35 beverages (reinforced), and also includes clinical nutrition products, as well as dietary supplements.

"Reinforcement" means that at least one extract of oregano or one or more volatile components thereof was added / added during the manufacture of the food / feed or beverage.

In humans, disorders connected with impaired or reduced neurotransmission include: depression, anxiety, irritability, unhappiness / discontent, sadness, dysphoria, obsessive-compulsive behavior, insomnia, and

40 abnormalities of biorhythms (altered circadian rhythms).

The compositions used in this invention can therefore be characterized as mood balancing agents, mood / vitality enhancers, stress relievers, anxiety reducers, stress reducers, relaxants, sleep enhancers, and biorhythm normalizers .

Numerous pharmaceutical compositions that are regulators of neurotransmitters have proven useful

45 in various disorders related to humor. Since it has been found that the extracts of this invention act using the same or similar biochemical mechanisms, it can be concluded that they are useful for similar conditions as absorption inhibitors.

Compounds that increase the levels of neurotransmitters in the brain and thus improve their transmission, can therefore exhibit antidepressant properties as well as beneficial effects on a variety of others.

50 mental disorders (Neurotransmitters. Drugs and Brain function, R.A. Webster (ed), John Wiley & Sons, New York, 2001, p. 187-211, 289-452, 477-498).

anxiety, and obsessive-compulsive behavior reducers. All of them improve, increase and reinforce physiological neurotransmission, especially in the central nervous system, and therefore relieve mental dysfunction.

Tricyclic antidepressant compounds (TCAs) such as imipramine, amitriptyline, and clomipramine, inhibit the

5 reabsorption of serotonin, norepinephrine and / or dopamine. They are generally considered among the most effective antidepressants available, but they have a number of disadvantages because they interact additionally with several brain receptors, e.g., with cholinergic receptors. And more importantly, eating disorders are dangerous since, if consumed in excessive doses, they frequently have acute cardiotoxicity.

10 Another class of antidepressant drugs are called SSRIs (selective serotonin reuptake inhibitors), which include fluoxetine, paroxetine, sertraline, citaloplam, and fluvoxamine, which block the serotonin transporter (SERT), a high affinity neurotransmitter transporter. dependent on sodium chloride that ends serotonergic neurotransmission by reabsorption of serotonin. Such drugs have proven effective in the treatment of depression and anxiety, and are usually better tolerated than eating disorders. These

15 medications are typically started at low doses and can be increased until they reach a therapeutic level. A common side effect is nausea. Other possible side effects include decreased appetite, dry mouth, sweating, infection, constipation, yawning, tremor, drowsiness and sexual dysfunction.

Likewise, systematic reviews and randomized placebo-controlled clinical trials (RCTs) indicate that some SSRIs (escitalopram, paroxetine and sertraline), SNRI venlafaxine, some benzodiazepines 20 (alprazolam and diazepam), the tricyclic antidepressant imipramine, and the partial agonist of 5-HT1A buspirone are all effective in acute treatment. In general, the effect of treatment is often moderate and symptoms reappear when the treatment period is interrupted. For this reason, a continuous long-term treatment or prevention with compounds that have minor side effects than SSRIs and can be followed for long periods of time could be favorable with respect to drug treatment. This can be achieved

25 by administration to people who need it from extracts of oregano or its volatile components in the form of dietary supplements.

For this reason, there is still a need for compounds for the method of improving disorders connected with impaired neurotransmission that do not exhibit the negative side effects of known antidepressants. Many patients are interested in alternative therapies with less or no side effects associated with

30 taking known drugs. Severe depression is a long-lasting and recurring disease, which is usually misdiagnosed. Additionally, many patients suffer from mild or moderately severe depression. Therefore, there is a growing interest in the development of compounds, as well as pharmaceutical and / or dietary compositions, that can be used to treat or prevent the development of mental illnesses / disorders such as depression, in people at risk, and to stabilize the humor.

Additionally, compounds that prevent the catabolism of neurotransmitters more widely by inhibition of monoaminooxidases (MAOs) A and B, called MAO inhibitors (MAOIs), exhibit antidepressant effects. MAOs catalyze the oxidation of neurotransmitters that contain amine groups such as serotonin, norepinephrine, and dopamine.

Additionally, neurotransmission modulators exert pleiotropic effects on mental and cognitive functions 40. In addition to depression, examples of other human conditions are listed below.

Generalized Anxiety Disorder (GAD) and Other Anxiety Disorders

It is well known that impaired neurotransmission, e.g. Low levels of neurotransmitters, is connected with various diseases and mental conditions such as depression and generalized anxiety disorders (GAD). Patients often suffer, either as a comorbidity to depression or in isolation, from generalized anxiety syndrome or generalized anxiety disorder. GAD is a highly prevalent anxiety condition and chronic disease in primary care (~ 10% of patients) (Wittchen et al, 2005 Eur. Neuropsychopharm. 15: 357-376). Patients are presented to their primary care physician with multiple physical symptoms. GAD is characterized by chronic tension, and anxiety and anxious tension (> 6 months), which are disabling and uncontrollable, and are accompanied by a characteristic hypervigilance syndrome (including 50 restlessness, muscle tension, and sleep problems). If left untreated, GAD follows a fluctuating chronic use and tends to become more serious with age. GAD patients suffer from depression sub-syndrome. GAD patients are classified as frequent users with the maximum direct and indirect global health burden of all anxiety and depressive disorders. Despite the high incidence of GAD, few patients are diagnosed, treated with medication, or referred to the psychiatrist; simple diagnostic instruments are required

55 to assist in the recognition and monitoring of patients. Regardless of the specific diagnosis, doctors require effective treatments for GAD symptoms. SSRIs such as paroxetine are effective for the treatment of GAD [Stocchi et al., 2003 J Clin Psychiatry 63 (3): 250.]

Several SSRIs have been approved by the FDA for the treatment of anxiety disorders:

fluoxetine -OCD, PD sertraline -OCD, PD, PTSD, SAD paroxetine -OCD, PD, SAD, GAD, PTSD

5 escitalopram -GAD fluvoxamine -OCD

[OCD: obsessive-compulsive disorder, PD: panic disorder, PTSD: post-traumatic stress disorder, SAD: social anxiety disorder, GAD: generalized anxiety disorder]

Aggression

10 Pathological impulsive aggressiveness can be associated with lower serotonergic innervation in the anterior cingulate cortex, as demonstrated using PET, where it has been shown that [11C] McN 5652 fixation was significantly reduced in this brain region [Frankle et al. 2005 Am. J. Psych., 162: 915-923]. In this regard, it should also be noted that serotonin dysfunction has been correlated with impulsive and violent criminal behaviors, alcohol abuse and suicide attempts, having also been reported in children

15 hospitalized for aggressive behavior.

Tryptophan dietary impoverishment has been shown to result in an increase in aggressive responses, in a laboratory situation, while the increase in tryptophan resulted in a decrease in aggressive responses. Thus, presumably, the decrease in serotonin is linked to an increase in aggression [Marsh et al. 2002, Neuropsychopharm 26: 660-671].

20 Attention deficit hyperactivity disorder (ADHD)

A number of antidepressants that affect the reabsorption of one or more of the monoamines are also effective in the treatment of ADHD and constitute a good alternative to commonly used stimulant medications: (1) tricyclic antidepressants (TCAs), such as imipramine, desipramine and amitriptyline, whose mechanism of action includes blocking norepinephrine and serotonin reabsorption and regulation

Decreasing β-adrenergic receptors; (2) dual reabsorption inhibitors, such as bupropion (dopamine / noradrenaline reuptake inhibitor) and venlafaxine (SNRI); (3) simple reabsorption inhibitors, such as atomoxetine and tomoxetine (blockade of norepinephrine transporters of the prefrontal presynaptic cortex) [Greydanus 2005. Ind. J. Ped., 72: 953-960; Chouinard 2005 J. Psych. & Neuro., 31 (3): 168-176].

30 Disturbances of Circadian Rhythms

Mood disorders and occupational stress can lead to disturbances in circadian rhythms (so-called biorhythms). These conditions are often chronic and persistent. Also, the dysregulation of circadian rhythms induced by long-haul flights (jet-lag), as well as by work in shifts, can cause similar symptoms and distress. Therefore, the use of dietary supplements to maintain the normal circadian rhythm

35 (to which an animal or human is habituated), and / or to relieve and prevent symptoms associated with a disturbed circadian rhythm, such as impaired cognitive function and memory, and mental and physical fatigue, is justified for improve the overall quality of life and increase the vital energy of a person who is in need of it.

Sleep Disorder, Insomnia, and Chronic Fatigue Syndrome

40 Sleep and depression are closely linked. EEG sleep abnormalities are common in depression, and insomnia can lead to depression. Sleep disturbances affect other biological rhythms involved in depression (Vogel et al. 1990 Neurosci Biobehav Rev 14: 49-63; Mc Carley 1982. Am J Psych 139: 565-570). Antidepressants can improve sleep continuity, reduce sleep with rapid eye movement (REM) and prolong slow wave sleep (SWS) (Sraner et al 2006 in Clinical Pharmacology of Sleep.

45 S.R. Pandi-Perumal et al (editors). Birkhäuser, Basel, pp. 103-124.

TCAs are commonly used to treat chronic fatigue syndrome. TCAs are believed to promote stage 4, sleep without rapid eye movement, [Craig et al 2002. Am. Fam. Physician, 65: 10831090].

Obsessive-Compulsive Disorder (OCD)

50 Improved neurotransmission in 5-HT2 receptors may be involved in the therapeutic action of SSRIs in the OCD; hyperactivity in the neuronal loop between the orbitofrontal cortex, the caudate nucleus head and the thalamus can be attenuated by SSRIs, due to increased activation of 5-HT2 inhibitor receptors in the orbitofrontal cortex [Blier et al 2001 J. Psych & Neuro. 26 (1): 37-43]. SSRIs, such as zimeldine, fluoroxetine and sertraline, have proven effective in the treatment of OCD [Chouinard, 2005 J. Psych. & Neuro. 31 (3): 168-176].

Pain

Antidepressants with different mechanisms of action may also be effective in the treatment of pain.

5 For example, amitriptyline and mianserin, which are potent 5-HT2 antagonists, are used for chronic pain control [Blier et al 2001. J.. Psych. & Neuro. 26 (1): 37-43]. The norepinephrine / serotonin / dopamine reabsorption inhibitor, duloxetine, is effective in the treatment of neuropathic pain associated with diabetic peripheral neuropathy [Chouinard, 2005 J. Psych. & Neuro. 31 (3): 168-176]. This type of pain is different from that associated with inflammation. In accordance with this invention, pain is reduced using a mechanism

10 different from the decrease in inflammation.

As an antidepressant, oregano extracts and their volatile components, as well as compositions / medications that contain them, can be used in methods of mental improvement, behavior, and emotional / affective, neurotic, neurodegenerative, food-related and food-related disorders. stress such as vg unipolar depression, bipolar depression, acute depression, chronic depression, subchronic depression, dysthymia, 15 postpartum depression, dysphoria / premenstrual syndrome (PMS) climacteric depressive symptoms, aggression, attention deficit disorders (ADS), social anxiety disorders, affective disorders seasonal, generalized anxiety / anxiety disorders (GAD), fibromyalgia syndrome, chronic fatigue, sleep disorders (insomnia), post-traumatic stress disorders, panic disorders, obsessive-compulsive disorders, restless legs syndrome, nervousness, migraine / primary headaches and pain in general, emesis, bulimia, anorexia nervosa,

20 disorder of excesses in the food, gastrointestinal disorders, syndrome of exhaustion, irritability and tiredness.

As an antidepressant, oregano extracts and their volatile components, as well as the compositions / medications containing them, can also be used for the manufacture of compositions for primary and secondary prevention and / or the treatment of neurocognitive impairment. Additionally, they are also effective in the treatment of depressive symptoms or other symptoms related to disturbed neurotransmission that appear as comorbidity in chronic diseases, e.g. cardiovascular diseases, attacks, cancer, Alzheimer's disease and Parkinson's disease. In accordance with this invention, oregano extracts and their volatile components, as well as the compositions / medications containing them, are not used for the treatment of cardiovascular diseases, strokes, cancer, Alzheimer's disease and Parkinson's disease proper, but to treat depression caused by these

30 diseases

Oregano extracts or their volatile components can be used for the manufacture of medications for the treatment of a disorder related to impaired neurotransmission. They can be additionally used for the manufacture of compositions for use as mood balancing agents, mood / vitality enhancers, stress relievers, condition enhancers, anxiety reducers,

35 stress reducers, sadness reducers, unhappiness / discontent reducers, irritability reducers, dysphoria reducers, obsessive-compulsive behavior reducers, relaxers, sleep improvers and / or insomnia relievers.

"Mood enhancer", "vitality enhancer" or "emotional well-being booster" means that the mood or vitality of a person treated with it is improved, that increases their self-esteem and / or that negative ideas and / or

40 negative tension, sadness, unhappiness and discontent, irritability and dysphoria are reduced. It also means that emotions are balanced and / or that general well-being, especially mental, and vitality are improved or maintained, and that the risk of mood fluctuations is reduced, that positive mood is maintained, and that one is Feel full of energy and motivated.

"Stress reducer, sadness reducer, unhappiness / discontent reducer, irritability reducer,

45 dysphoria reducer "means that tension (chronic) and worry are reduced or relieved. Hypervigilance syndrome, including restlessness and muscle tension, is reduced or relieved as well. Social and other phobias are They also resolve at least partially.In general, the social environment feels less threatening.The person is emotionally relaxed, experiences comfort and enjoys company and contact with other people.In general, the person feels full of energy and motivated to perform the tasks

50 daily

A "relaxant" acts by completely or partially correcting the circadian rhythm (biorhythm) of a person who has been disturbed due to jet-lag or shift work. A relaxant will at least partially prevent or cancel out the symptoms associated with such disturbances, that is, the deterioration of cognitive function and memory, mental and physical fatigue, and will improve overall the quality of life and vital energy. Thus, oregano extracts or one or more of their

55 volatile components can also be used to prevent and / or nullify the deterioration of cognitive function and memory, to prevent and / or cancel mental and physical fatigue, and to improve the overall quality of life and vital energy.

A further embodiment of the present invention relates to the use of oregano extracts or their volatile components and to the use of compositions containing them as "condition improvers", that is as means to reduce irritability and fatigue, to reduce, prevent or relieve physical and mental fatigue, to promote calm sleep, that is, to act against insomnia and sleep disorders and improve sleep, and to increase energy in more general terms, especially to increase brain energy production , in sick or normal health individuals. In addition, such "state enhancers" can be used to improve cognition in general, and especially for maintenance or improvement of attention and concentration, memory and ability to remember, learning ability, language processing, of problem solving and intellectual functioning; for short-term memory improvement; to increase the state of

10 mental alert; to increase the ability to concentrate and mental acuity, to intensify mental vigilance; for reduction of mental fatigue; for the support of cognitive well-being, for the maintenance of a balanced cognitive function, for the regulation of hunger and satiety, and for the regulation of motor activity.

Thus, a preferred aspect of the invention relates to the use of oregano extracts and their volatile components as mood balancing agents and / or stress relievers.

In a further preferred embodiment of the present invention, oregano extracts or their volatile components are used to maintain circadian rhythms in humans, for relief and / or for prevention of symptoms associated with a disturbed circadian rhythm in humans. Thus, humor is stabilized and an emotional balance is achieved to cope with the stress of daily life and maintain physical and psychological efficiency.

20 Additionally, the symptoms associated with a disturbed circadian rhythm, such as impairment of cognitive function and memory, as well as mental and physical fatigue, are relieved and / or prevented, thereby improving the overall quality of life. These people also benefit from the maintenance of vital energy. Also, the dysregulation of circadian rhythms induced by long-haul flights (jet-lag), as well as by shift work and the symptoms associated with it, are alleviated and / or prevented.

Another preferred aspect of the invention relates to the use of oregano extracts or their volatile components for the manufacture of a composition, for use as an antidepressant.

In the context of this invention, "treatment" also encompasses co-treatment as well as prevention. "Prevention" may be the prevention of the first occurrence (primary prevention) or the prevention of a recurrence (secondary prevention). Prevention also means that the risk of suffering impaired neurotransmission, or of

30 unbalanced mood or stress is reduced. The term "prevention" especially includes reducing the risk or incidence of the development of certain symptoms, especially associated with a disturbed circadian rhythm.

In another embodiment, an effective dose of oregano extracts or their volatile components can be used especially for maintaining mental well-being, for maintaining a balanced cognitive function, for contributing to the maintenance of a positive mood, relaxation, and for strengthening cognitive well-being.

The extracts or their volatile components of this invention improve, increase and reinforce physiological neurotransmission, especially in the central nervous system, and therefore can relieve mental dysfunction.

Another additional aspect of this invention is a method for prevention or treatment of disorders connected with impaired neurotransmission in humans by increasing the plasma level of carvacrol to at least 10 ng / ml in humans, preferably by increasing the level of plasma carvacrol up to within. from the range of 10 ng / ml to

40 51,000 ng / ml in humans. The plasma level can be measured as described in the examples.

VETERINARY USES

Another aspect of this invention are veterinary uses of oregano extract and / or its volatile components. Animals can exhibit adverse behavioral and / or physiological reactions to stressful situations. For example, animals raised in large-scale production environments, or that are transported, can

45 have a decrease in the quantity or quality of meat or milk. Stressed poultry can resort to plucking, reduced egg laying and cannibalism. Many animals can become aggressive or exhibit obsessive-compulsive behaviors. The extracts and volatile components of this invention, by acting as neurotransmitters, can alleviate these undesirable behaviors and physiology in animals.

In a preferred embodiment of the present invention, oregano extracts or their volatile components are administered for stress prevention in farm animals, in livestock rearing for large-scale production, during transport to the slaughterhouse and / or for prevention. of the loss of meat quality in these animals during transport to the slaughterhouse.

In another preferred embodiment of the present invention, oregano extracts or their volatile components are

55 administered to pets or pets for reduction of stress, tension and aggressiveness and compulsive behavior exhibited in stressful conditions, such as separation, change or loss of the owner, during the separation on holidays and the permanence in so-called "animal hotels", and maintenance in shelters or animal shelters.

Especially companion animals and farm animals may be in conditions of need for improvement or increase in neurotransmission. Such conditions are presented e.g. after capture or the

5 transport or with accommodation, when animals develop analogous disorders and suffer anguish or aggressiveness, or exhibit stereotypical behaviors, or anxiety, tension, sadness, unhappiness / discontent and irritability, dysphoria and obsessive-compulsive disorder.

Thus, oregano extracts or their volatile components, and the preferences set forth above, can generally be used as antidepressants for animals including humans, preferably for

10 humans and other mammals, particularly for pets and farm animals.

Another embodiment of this invention is a method for reducing stress in farm animals in raising cattle for large-scale production, during transport to the slaughterhouse and / or for preventing the loss of meat quality of said farm animals. during transport to the slaughterhouse, which comprises administering an effective dose of an oregano extract or one or more of its volatile components to farm animals that are

15 find themselves in need of it. Farm animals are preferably poultry, cattle, sheep, goats and pigs.

In another preferred embodiment of the present invention, oregano extracts or their volatile components are administered to poultry to prevent plucking and cannibalism that result in e.g. losses of meat quality and egg production. Thus, another aspect of this invention

20 is a method for preventing the loss of egg production, plucking and cannibalism and loss of meat quality due to transport stress among poultry, which comprises administering an effective dose of a Oregano extract or one of its volatile components to poultry that are in need of it.

Another aspect of this invention is a method for prevention and / or relief of fish farm stress comprising the

25 step of administering an effective dose of an extract of oregano or one or more of its volatile components to animals that are in need thereof, where the animals are fish or shrimp.

Another additional aspect of this invention is a method for reducing stress, tension, aggressiveness and / or compulsive behavior in companion animals that are under stressful conditions, such as separation, change or loss of the owner, during the separation of holidays and maintenance on

30 called "animal hotels", as well as maintenance in shelters, and other conditions of dense breeding and reproduction, which includes the step of administering an effective dose of an extract of oregano or one or more of its volatile components to pets that are in need of it, especially cats and dogs that are in need of it.

Another additional aspect of this invention is a method for prevention and / or reduction of associated symptoms.

35 with stressful conditions in animals used for the skin industry, preferably for minks, foxes and / or hares.

In the case of animals, oregano extracts or their volatile components are preferably administered as reinforced feed or reinforced beverages (e.g. as an addition to drinking water).

Oregano extracts or their volatile components

40 The extracts of oregano can be of any origin of a plant (enter plant or parts thereof) belonging to the genera Origanum such as Origanum vulgare and Thymus such as Thymus vulgaris in the form of a concentrate of extractable compounds, especially volatile compounds. Additional examples of plants of the genus Origanum encompassed by the term "oregano" are O. majorana, O. dictamus, O. creticum, O. majoricum, O. aureum, O. compactus, O. syriaca, O. tyt-thantum, O heracleoticum, O. smyrnaeum and O. virens. Examples

An additional 45 plants of the genus Thymus encompassed by the term "oregano" are T. herbus-barona, T. citriodorus, T. mastichiana, T. pulegioides, T. serpyllum, T. pallasianus and T. praecox. The concentrate may also contain solvents used for extraction, may be exempt from them or may be transferred to specific carrier materials. Extracts can be obtained according to methods well known in the art, e.g., by (one) solvent extraction such as methanol, ethanol, ethyl acetate, diethyl ether, n-hexane, chloride

50 of methylene, or with supercritical fluids such as carbon dioxide (pure or in admixture with other solvents such as alcohols) or dinitrogen oxide, (b) hydro-distillation to obtain essential oils or (c) extraction / distillation with hot gases such as nitrogen.

Preferably, oregano extracts are obtained which are obtained by an extraction with the use of supercritical carbon dioxide. Such extracts have the advantage that they do not contain any amount of organic solvents, proteins or heavy metals. If desired, an extraction with supercritical carbon dioxide is followed

by a second extraction step with supercritical fluid CO2 to remove waxes and selectively enrich volatile materials.

The extracts of oregano or its volatile components may be of natural, synthetic or mixed origin (i.e. partially natural and partially synthetic), that is, they can, apart from being obtained by extracting

5 plants and fractionation, chemically synthesized and, if desired, mixed with each other in any desired quantities. They can be prepared and used in any desired purities and concentrations, e.g., as solutions containing them in concentrations as low as, e.g., 10% (w / w) or less, or up to about 100% (w / w).

Oregano extracts containing a high proportion of at least one of its components are preferred

10 volatile More preferred are oregano extracts that contain at least a total of 70% by weight volatile components as mentioned above, based on the total weight of the extract. Completely natural oregano extracts can be reinforced with at least one specific volatile component thereof.

Preferred oregano extracts in the context of the present invention are those in which:

oregano extract comprises at least 30% by weight of carvacrol,

The oregano extract comprises at least 50% by weight of carvacrol, more preferably those in which the oregano extract comprises at least 60% by weight of carvacrol, and most preferably those in which the oregano extract comprises at least 65 % by weight of carvacrol, based on the weight of oregano extract.

Oregano extracts and oregano extracts comprising thimoquinone in an amount are also preferred.

20 in the range of 0 to 30% by weight, preferably wherein the oregano extract comprises at least 1% by weight of thimoquinone, more preferably where the oregano extract comprises at least 2% by weight of thimoquinone, even more preferably where the oregano extract comprises at least 5% by weight of thimoquinone and most preferably where the oregano extract comprises thimoquinone in a range of 5 to 30% in

25 weight, based on the weight of oregano extract.

Other preferred oregano extracts are those in which the oregano extract comprises at least 50% in weight of carvacrol and from 0 to 25% by weight of thimoquinone, preferably wherein the oregano extract comprises at least 50% by weight of carvacrol and at least 1% in thimoquinone weight;

30 more preferably wherein the oregano extract comprises at least 55% by weight of carvacrol and at least 2% by weight of thimoquinone, even more preferably where the oregano extract comprises at least 60% by weight of carvacrol and at least 5 % by weight of thimoquinone, and most preferably wherein the oregano extract comprises at least 65% by weight of carvacrol and thimoquinone

35 in a range of 5 to 25% by weight, based on the weight of oregano extract.

Methods in which simple volatile components or their mixtures are used are also preferred, wherein the volatile components are selected from the group consisting of carvacrol, thimoquinone, p-cimeno, thimoquinol, limonene, linalool, borneol, 4-terpineol, thymol and karyophylene . Preferably, the volatile compounds are selected from the group consisting of carvacrol, thimoquinone and p-cimeno, more preferably wherein the components

Volatiles are carvacrol and / or thimoquinone, and most preferably where the volatile component is carvacrol.

Even more preferred are oregano extracts that do not contain: a hydrophilic extract, an essential amount of one of the following constituents: rosmarinic acid, eugenol, eugenol salts, eugenol isomers, yeast cell walls or 1-piperoylpiperidine. An "essential amount" as used herein the total amount of any of these ingredients, if present at all, is preferably less than 0.5% by weight, plus

Preferably less than 0.2% by weight, even more preferably less than 0.1% by weight, based on the total weight of said oregano extract or oregano material or the volatile component (s).

Additionally, certain combinations of plant extracts are not preferred in this invention, such as the combination of oregano extract and: Agaricus blazei, nettle, artichoke, Crataegus, Leonurus, common yarrow, mistletoe, Crataeg, Herba viola

50 tricolor, Scutellariae baicalensis, turmeric, gold thread, barberry.

The composition of the present invention is preferably in the form of nutritional composition, such as reinforced food, reinforced feed, or reinforced beverages, or in the form of food / liquid reinforced feed for animals, including humans.

The dietary and pharmaceutical compositions according to the present invention can be found in

55 any galenic form that is suitable for administration to the animal body including the human body, especially in any form that is conventional for oral administration, e.g. in solid form, such as (additives / supplements for) food or feed, food or feed premix, reinforced food or feed, tablets, pills, granules, dragees, capsules, and effervescent formulations such as powders and tablets, or in liquid form such as solutions, emulsions or suspensions such as eg drinks, pastes and oily suspensions. The pastes can be encapsulated in hard or soft pod capsules, in which case the capsules

5 present e.g. a matrix of gelatin (fish, pork, poultry, cow), plant proteins or ligninsulfonate. Examples of other forms of application are forms for transdermal, parenteral or injectable administration. Dietary and pharmaceutical compositions may be in the form of controlled release (delayed) formulations. The compositions of the present invention are not administered nasally.

The dietary compositions according to the present invention may additionally contain hydrocolloids.

10 protectors (such as gums, proteins, modified starches), binders, film-forming agents, encapsulating agents / materials, wall / sheath materials, matrix compounds, coatings, emulsifiers, surfactants, solubilizing agents (oils, fats, waxes , lecithins, etc.), absorbents, carriers, fillers, co-compounds, dispersing agents, wetting agents, processing aids (solvents), fluidizing agents, taste masking agents, weight-increasing agents, gelling agents,

15 gel-forming agents, antioxidants and antimicrobials.

Examples of foods are cereal bars, dairy products, such as yogurts, and pastries, such as cakes and cookies. Examples of reinforced food are cereal bars, and baked goods, such as bread, rolls, donuts, cakes and cookies. Examples of dietary supplements are tablets, pills, granules, dragees, capsules and effervescent formulations, in the form of non-alcoholic beverages, such as

20 soft drinks, fruit juices, lemonades, drinks consisting almost exclusively of water, teas and milk drinks, in the form of liquid food, such as soups and dairy products (muesli drinks).

Drinks include non-alcoholic beverages and alcoholic beverages as well as liquid preparations to add to drinking water and liquid foods. Non-alcoholic beverages are e.g. soft drinks, sports drinks, fruit juices, vegetable juices, e.g. tomato juice), lemonades, teas and milk drinks. Liquid foods are

25 v.g. soups and dairy products (e.g. muesli drinks).

In addition to at least one extract of oregano or one of its volatile components, the pharmaceutical compositions according to the present invention may additionally contain conventional pharmaceutical additives and adjuvants, excipients or diluents, including, but not limited to, water, gelatin of any origin, vegetable gums, ligninsulfonate, talc, sugars, starch, gum arabic, vegetable oils,

30 polyalkylene glycols, flavoring agents, preservatives, stabilizers, emulsifying agents, buffers, lubricants, colorants, wetting agents, fillers, and the like. The carrier material may be an organic or inorganic inert carrier material suitable for oral / parenteral / injectable administration.

For humans, an adequate daily dose of oregano extracts or their volatile components for the purposes of the present invention may be within the range of 0.001 mg per kg of weight.

35 body at approximately 100 mg per kg body weight per day. A daily dose of about 0.01 to about 10 mg per kg body weight is more preferred, and a daily dose of about 0.05 to 5.0 mg per kg body weight is especially preferred.

In unit preparations in solid form for humans, the oregano extract or its volatile components is / are suitably present in an amount ranging from about 0.1 mg to about 40 1000 mg, preferably from about 1 mg to about 500 mg per unit dose. For relief of symptoms associated with conditions such as those mentioned herein, oregano extract or any of its volatile components is taken once or twice a day along with a meal for at least a week and up to 6-12 months For prevention of the appearance of symptoms associated with conditions such as those mentioned herein, and for maintaining a relaxed state in general, it is

45 adequate consumption on a regular basis.

In dietary compositions, especially in foods and beverages for humans, oregano extract or its volatile components is / are conveniently present in an amount ranging from about 0.0001 (1 mg / kg) to about 5% by weight (50 g / kg), preferably from about 0.001% (10 mg / kg) to about 1% by weight (10 g / kg), more preferably from about 0.01 (100

50 mg / kg) at approximately 0.5% by weight (5 g / kg) based on the total weight of the food or drink. To alleviate the symptoms associated with conditions such as those defined above, the food product is taken once or twice a day for at least 1 to 3 weeks or on a regular basis, that is at least once a day.

In foods and beverages in a preferred embodiment of the invention, the amount of oregano extract or its volatile components is / is 10 to 30 mg per serving, i.e. 120 mg per kg of food or drink. The product

The food is taken once or twice a day for at least 1 to 3 weeks or preferably on a regular basis at least once a day.

For animals excluding humans, a suitable daily dose of an extract of oregano or its volatile components, for the purposes of the present invention may be within the range from 0.001 mg / kg body weight to approximately 1000 mg per kg of body weight per day. A daily dose of about 0.1 mg to about 500 mg per kg of body weight is more preferred, and a daily dose of about 1 mg to 100 mg per kg of body weight is especially preferred. To prevent and reduce the symptoms associated with stressful conditions, such as the raising of cattle for large-scale production and for industrial breeding or fish farming, the product containing oregano extract

or his / her volatile components is administered throughout the life of the animal until its conduction to the slaughterhouse. Especially in the case where farm animals, such as poultry, cattle, sheep, goats and pigs, especially cattle and pigs, are transported to the slaughterhouse, they should be administered at a daily dose of approximately 3 to 800 mg / kg body weight of the extract and / or its volatile components, preferably during transport, more preferably at least 3 days before transport and during transport.

For pets, under stressful conditions such as on farms or animal shelters or pet stores, the product should be administered at least 1-3 weeks, or throughout the entire breeding period. In conditions of short-term stress, such as a separation during holidays, maintenance in "hotels for animals", visits to or stay in veterinary clinics, the product can be administered at least 3 days, and preferably 7 days, before the stressful event.

The invention is further illustrated by the following examples.

Examples

Example 1: Preparation of two extracts of O. vulgare

In the following examples, "-se" refers to phenol-type oregano extract 1, obtained from, and "-to" refers to terpineol-type oregano extract 2, both obtained from Flavex, Germany.

Dry leaves of O. vulgare were ground and extracted with supercritical carbon dioxide. The extraction parameters were as follows: temperature of 45 ° C; working pressure: 300 bar (-to) or 100 bar (-se); 17 kg (-to) and 15 kg (-se) of carbon dioxide were needed for 1 kg of plant material; The extracts were obtained in the separator by throttling the pressure at 60 bar at 30 ° C.

25 kg (-to) or 50 kg (-se) of plant material produced 1 kg of extract respectively.

Extract 1 had the following composition (analyzed by Gas Chromatography)

The total essential oil content was 83% (the remaining parts are vegetable waxes)

Volatile components: 0.2% terpinen, 2.6% cimeno, 1.5% 4-terpineol, thymquinone, 23%, 0.3% thymol, 62% carvacrol, 1.5% karyophylene.

Extract 2 (RV141-23) contained 80-90% essential oil with a high content of terpineols that included trans-beta-terpineol 35-50%, cis-beta-terpineol 5-10%, 4-terpineol 8-12 %, and a relatively small amount of phenols that included 5-12% thymol, carvacrol <1%, based on the total content of essential oils.

Example 2: Preparation of oregano extract 3

Origanum vulgare sheets were extracted with a solvent mixture consisting of methyl-tert-butyl ether and methanol with the 9: 1 volume ratio.

Example 3: Preparation and composition of oregano extract 4 Flavex

Oregano leaves were prepared according to Example 1, pure extract. Due to a different collection lot, the composition was slightly different from extract 1.

The total essential oil content was 89.5% (the remaining parts are vegetable waxes)

Volatile components: cimeno 7.8%, 4-terpineol 1.2%, thimoquinone, 12.1%, thymol 0.22%, carvacrol 68.5%, cariophylene 1.6%, limonene 0.1%, linalol 0 , 77%, borneol 2.6%.

Example 4: Detailed composition of oregano extracts

Extracts were prepared by steam distillation of oregano leaves.

The composition was as follows:

GC / MS 10 analysis method

GC / MS

GC / MS

GC / MS

Carvacrol

Thimoquinone Timol

p / p

p / p

p / p

Extract 5 (spice Oregano dragon)

61.6 - 9.0

Extract 6 (Yafa herbs / Morocco)

30.7 - 21.6

Extract 7 (Aysun / Derial, Turkey)

64.9 - 0.3

Example 5: Inhibition of serotonin absorption by oregano extracts

HEK-293 cells stably expressing the human serotonin resorption transporter (hSERT) were obtained from R. Blakely, Vanderbilt University, USA. The cells were routinely grown in Dulbecco's Modified Eagle's Medium, purchased from Bioconcept, Allschwil, Switzerland, which contained 10% serum of fetal calf, penicillin, streptomycin, L-glutamine and the G418 antibiotic, and underwent passes by trypsination. One day before the test, the cells were seeded in the above mentioned medium. Immediately before the test, the medium was replaced by Krebs-Ringer bicarbonate buffer, purchased from Sigma Chemicals Ltd., supplemented with 35 μM pargiline, 2.2 mM CaCl 2, 1 mM ascorbic acid and N-2-hydroxyethylpiperazine-N ' -2 5 mM ethanesulfonic acid ("Hepes" buffer). Serotonin absorption in cells was determined by the addition of

20 radiolabeled serotonin (3H) (Amersham Biosciences GE Healthcare, Slough, UK) at a concentration of 20 nM, and incubation for 30 minutes at room temperature. After removal of the unincorporated label by gentle washing 3 times with the above buffer, the incorporated serotonin was quantified by counting by liquid scintillation.

Serotonin uptake by the transporter was inhibited by oregano extract in a dependent manner

25 of the dose. The IC50 values measured for the inhibition of serotonin uptake by 3 oregano extracts are shown in Table 1.

Table 1: IC50 values measured for inhibition of serotonin absorption in HEK-293 transfected cells by oregano extracts 1, 2 and 3, and their volatile components thymol, carvacrol and thimoquinol. Data are shown as mean ± s.e.m., where IC50 is expressed as μM for simple compounds and as μg / ml for

30 extracts

Substance

IC50 [μM or μg / ml] for absorption of tritiated serotonin

Oregano Extract 1

3.2 μg / ml ± 1.4

Oregano Extract 2

15.31 μg / ml ± 5.3

Oregano Extract 3

66.3 μg / ml ± 3.0

Oregano Extract 4

1.95 μg / ml ± 0.94 (n = 3)

Oregano Extract 5

7.8 μg / ml

Oregano Extract 6

5.8 μg / ml

Oregano Extract 7

4.1 μg / ml

Timol

5.0 μM ± 0.4

Carvacrol

9.5 μM

Thimoquinol

26.6 μM

Example 6: Inhibition of Monoaminooxidases by oregano extract 1

Organic p-tyramine or benzylamine amines were used as substrates for the enzymes Monoamine Oxidase A (MAO-A) and B (MAO-B), respectively. The hydrogen peroxide (H2O2) produced by this reaction was quantified by reaction with vanillinic acid, catalyzed by horseradish peroxidase (HRP).

The reactions were carried out in polystyrene microtiter plates. The MAO enzymes (final concentration 2 U / ml) were mixed with p-tyramine (Sigma, final concentration 0.5 mM) or benzylamine (Sigma, final concentration 0.5 mM) if appropriate and the chromogenic solution (containing acid vanillin (Fluka), 4-aminoantipyrine (Fluka) and horseradish peroxidase (Sigma, final concentrations 0.25 mM, 0.125 mM and 1 U / ml,

5 respectively) in 0.2 M potassium phosphate buffer pH 7.6. The reactions were followed in a microtiter plate absorbance reader, e.g. Spectramax M5 (Molecular Devices Corporation). Absorbance readings were taken at 495 nm every 15 seconds for 40 minutes and the initial reaction rates were calculated by linear regression using SOFTmaxPro (Molecular Devices Corporation).

The effect of oregano extract 1 on monoamine oxidase enzymes was determined by its inclusion in the

10 assay in a concentration range between 0.03 and 100 μM for 10 minutes before and during substrate incubation. To determine the effect of the compounds on the portion of the reaction catalyzed with HRP, the MAO enzyme was replaced by H2O2 (Molecular Probes, 0.2 mM final concentration). Reactions containing MAO-A and -B were both inhibited by oregano extract 1 in a dose-dependent manner, while the control reaction remained unaffected. IC50 values measured for inhibition of the activity of

Monoamine oxidase by oregano extract 1 are shown in Table 2.

Table 2: IC50 values measured for inhibition of Monoamine-Oxidase-A and -B enzymes by oregano extract

1. Data are shown as the mean ± s.e.m.

Substance

IC50 [μg / ml] for MAO-A inhibition IC50 [mg / ml] for MAO-B inhibition

Oregano Extract 1

2.7 μg / ml ± 0.8 s.e.m. n = 2 13.4 μg / ml ± 3.6 s.e.m. n = 2

Example 7: Effect of oregano extract 1 on the Primary Observation Test (Irwin) in the mouse

20 The method, which detects the first toxic dose, the active dose range and the main effects of a test substance on behavior and physiological function, follows that described by Irwin (Irwin S. 1968 Psychopharma. 13: 222-257 ).

The test substance was administered to mice and the animals were observed in simultaneous comparison with a control group to which vehicle was administered (non-blind conditions). 3 groups treated with the same were compared

25 control group at any time. All animals within a treatment group were observed simultaneously.

Behavioral modifications, physiological and neurotoxicity symptoms, pupil diameter and rectal temperature were recorded according to a standardized observation grid derived from Irwin's. The grill contains the following sections: lethality *, convulsions *, tremor *, Straub *, sedation, excitation,

30 abnormal gait * (wobble, tiptoe), jumps *, motor uncoordination *, contortions *, piloerection *, stereotypes * (sniffing, chewing, head movements), head shaking *, scratching *, breathing *, aggressiveness *, tremor, reactivity to touch, muscle tone, loss of straightening reflex, ptosis, exophthalmos, loss of grip, akinesia, catalepsy, loss of traction, loss of corneal reflex, analgesia, defecation, salivation, lacrimation, pupil diameter (Unit = 1/45 mm) and rectal temperature.

35 Observations were made 15, 30, 60, 120 and 180 minutes after administration of the test substance and also 24 hours later. Marked symptoms (*) were observed continuously from 0 to 15 minutes after administration. Five mice were studied per group. Oregano extract 4 was solubilized in 3% (v / v) DMSO, 3% (v / v) Tween 80 in saline (0.9% w / v NaCl) and injected into mice intraperitoneally (ip ).

40 Results

-

Oregano extract 1 to 3 mg / kg did not induce additional behavioral changes, compared to the vehicle.

-

At 10 mg / kg, it induced light sedation in 3 mice and reduced fear from 15 to 30 minutes in all 5 mice.

45 -A 30 mg / kg, extract of oregano 1 induced light sedation in 4 or 5 mice for 15 to 60 minutes, induced fear in 3 or 4 mice from 15 to 60 minutes and reduced muscle tone in 1 to 5 mice 30 to 60 minutes

-

At 100 mg / kg, oregano extract 1 induced sedation that was mild to pronounced in 4 or 5 mice from 15 to 180 minutes and light in 1 mouse at 24 hours. It reduced fear in 4 or 5 mice from 15 to 180 minutes, reduced reactivity to touch in 4 mice from 60 to 180 minutes and reduced muscle tone in 3 or 4 mice from 30 minutes to 24 hours .

Thus, oregano extract 1 exhibited a moderate and dose-dependent relaxing sedative effect, and reduced fear.

5 Example 8: Porsolt Swimming Test

The "Desperate Behavior Test" or "Porsolt Forced Swimming Test" is an animal model validated for depression (see Nagatsu, 2004 NeuroTox., 25: 11-20, and Porsolt et al., 1977 Arch. Int. Pharmacodyn 229: 327-336). It responds to the improvement of the transmission of several neurotransmitters that include serotonin, dopamine and norepinephrine.

10 The test, which detects antidepressant activity, was carried out as described by Porsolt et al. Mice that are forced to swim in a situation from which they cannot escape quickly become immobile. Antidepressants reduce the duration of immobility.

The mice were individually placed in cylinders (height = 24 cm, diameter = 13 cm) containing 10 cm of water (22 ° C) from which they could not escape. The mice were put in the water for 6 minutes and the

15 duration of immobility during the last 4 minutes.

15 mice were studied for each of the 4 groups. The test was performed blindly, that is to say that the person conducting the experiment was different from the person injecting the mice and therefore did not know which of the 4 groups each mouse belonged to.

Oregano extract 1 was evaluated at 3 doses (10, 30 and 60 mg / kg), administered i.p. 30 minutes before the test, and it

20 compared with a control group, to which vehicle was administered in the same manner. The oregano extract 4 thus administered was dissolved in vehicle (saline solution containing 3% (v / v) of DMSO and 3% (v / v) of Tween® 80). Venlafaxine (16 mg / kg, i.p.), as a comparison substance, and imipramine (32 mg / kg, i.p.), were administered as the reference compound, under the same experimental conditions.

The data were analyzed by comparison of the groups treated with the control group using paired Student test 25, and are presented in Table 3.

Table 3: Reduction of "immobility time" with increasing concentration of oregano extract 1.

TREATMENT (mg / kg) i.p., -30 min

DURATION OF IMMOBILITY (s)

Mean ± s.e.m.

% Change from control

Vehicle

164.7 ± 7.6 - -

Oregano Extract 1 (10) Oregano Extract 1 (30) Oregano Extract 1 (60)

163.7 ± 10.3 137.1 ± 9.5 125.6 ± 6.5 -1% -17% -24% NS * ***

Venlafaxine (16)

80.1 ± 11.1 -51% ***

Imipramine (32)

49.7 ± 7.9 -70% ***

15 mice per group

Student t-test: NS = not significant; * = p <0.05; *** = p <0.001

30 Thus, oregano extract 1 significantly reduced the immobility time compared to the control group, by 17% and 24% in the intermediate and maximum dose groups, respectively. As a whole, a significant dose-dependent effect of oregano extract 1. Imipramine (32 mg / kg ip) and venlafaxine (16 mg / kg, ip), administered under the same experimental conditions, significantly reduced the behavior of immobility, compared to vehicle control (-70% and -51%, respectively, p <

35 0.001).

These results demonstrate that oregano extract 1 (60 mg / kg, i.p.) has a similar efficacy as the tricyclic antidepressant imipramine, and SNRI, venlafaxine, in its ability to significantly reduce behavior related to depression.

Example 9: Marble Burial Test as an Anxiety or Obsessive Compulsive Behavior Test40

Behavior 1979, 31: 299-306) or shock punctures (Pinel et al 1978, J. Comp. Physiol. Psych. 92: 708-712). The marble burial test was devised as a modification of that test. Poling et al. 1981 J. Exp. Anal. Behavior 1981, 35: 31-44) exposed rats to individual cages each containing 25 marbles, daily for 10 or 21 consecutive days. The number of buried marbles was counted, each day of the 10-day period, or 24 hours after exposure for 21 days. The authors stated that the burial of marbles was not determined by the novelty, or due to any harmful stimulus.

It has been reported that the burial behavior of marbles by mice is sensitive to a range of minor (eg diazepam) and major tranquilizers (eg haloperidol) (Broekkamp et al., 1986 Eur. J. Pharmacol. 126: 223-229) In addition to SSRIs (eg fluvoxamine, fluoxetine, citalopram), tricyclic antidepressants (eg imipramine, desipramine) and selective noradrenaline absorption inhibitors (eg reboxetine), at doses that do not induce sedation. The model may reflect anxiety-like behavior or obsessive-compulsive behavior (see De Boer et al, 2003 Eur. J. Pharma. 463: 145-161).

The method applied here follows that described by Broekkamp et al., 1986, supra. The mice (n = 15 per treatment group) were placed individually in transparent plastic cages (33 x 21 x 18 cm) with 5 cm of sawdust on the ground and 25 marbles (1 cm in diameter) grouped in the center of the cage. A second cage, turned upside down, served as a lid. The number of marbles covered by sawdust (at least two-thirds) was counted at the end of the 30-minute test period. The tests were performed by blind researchers to the drug treatment protocol.

Before the test, all the cages and marbles were "impregnated" leaving 10 naive mice in each cage for 15 minutes.

Oregano extract 1 to 10, 30 and 60 mg / kg, administered i.p. 30 minutes before the test, and it was compared with a vehicle control group. Oregano extract 1 was dissolved in a saline solution containing 3% (v / v) of DMSO and 3% (v / v) of Tween® 80 "vehicle." Vehicle was administered to the control group in the same way, while fluoxetine (32 mg / kg) was used as the reference substance, administered under the same experimental conditions. The data were analyzed by comparison of the groups treated with the vehicle control using unpaired Student t-tests.

Results:

Table 4: Effects of oregano extract 1 and fluoxetine on the marble burial test in mice

TREATMENT (mg / kg) i.p., -30 min

NUMBER OF CANICAS COVERED BY SERRIN

Mean ± s.e.m.

% Change from control

Vehicle

21.2 ± 1.6 -

Oregano Extract 1 (10) Oregano Extract 1 (30) Oregano Extract 1 (60)

12.7 ± 2.7 5.8 ± 2.6 6.9 ± 2.6 -40% * -73% *** -67% ***

Venlafaxine (16) Fluoxetine (32)

0.5 ± 0.3 1.4 ± 1.0 -98% *** -93% ***

15 mice per group

Student t-test: NS = not significant; * = p <0.05; *** = p <0.001

Oregano extract 1 (10, 30 and 60 mg / kg), administered i.p. 30 minutes before the test, the number of marbles covered was reduced in a dose-dependent manner, compared to the control vehicle control (40%, p <0.05, -73%, p <0.001 and -67%, p <0.001, respectively).

Fluoxetine (32 mg / kg ip) and venlafaxine (16 mg / kg, ip), administered under the same experimental conditions, practically nullified the burial of the marbles, compared to vehicle control (-93% and -98%, respectively, p <0.001).

These results demonstrate that oregano extract 1 has an efficacy similar to SSRI, fluoxetine, and SNRI, venlafaxine, in its ability to significantly reduce anxiety / obsessive-compulsive behavior.

Example 10: Effect of Oregano Extract 1 in the Dark-Illuminated Cage Test in the Mouse

The method, which detects anxiolytic activity, follows that described by Crawley, 1981 Pharmacol. Biochem Behav., 15: 695-699. Anxiolytics increase the time spent in the lit compartment.

The animals were placed in the illuminated compartment of a 2-compartment cage, with one half illuminated and open (25 x 27 x 27 cm) and the other half dark and closed (20 x 27 x 27 cm). The time spent in each compartment, as well as the number of times the animal crosses from one side to the other, is recorded during a 3-minute test. 15 mice were studied per group. The test was performed blindly.

Oregano extract 1 was evaluated at 3 doses (10, 30 and 60 mg / kg), administered i.p. 30 minutes before the test, and it was compared with a vehicle control. Oregano extract 1 was dissolved in a saline solution containing 3% (v / v) of DMSO and 3% (v / v) of Tween® 80 ("vehicle"). Vehicle was administered to the control group, used

10 venlafaxine (16 mg / kg i.p.) as a comparison substance and clobazam (16 mg / kg, i.p.) was used as the reference substance, all of which were administered i.p., 30 minutes before the test.

Table 5: Test result of the Dark-Illuminated Cage in the mouse

TREATMENT (mg / kg) i.p. -30 min

PAST TIME IN THE ILLUMINATED COMPARTMENT (s) NUMBER OF CROSSINGS

mean ± s.e.m.

% change from control  mean ± s.e.m. % change from control

Vehicle

55.3 ± 6.3 - 4.7 ± 1.1 -

Oregano Extract 1 (10) Oregano Extract 1 (30) Oregano Extract 1 (60)

70.7 ± 7.5 56.8 ± 5.8 74.9 ± 7.2 NS + 28% NS + 3% NS + 35% 7.1 ± 0.9 3.2 ± 0.7 3.5 ± 0.6 NS + 51% NS -32% NS -26%

Venlafaxine (16)

55.2 ± 9.3 NS 0% 4.4 ± 0.9 NS -6%

Clobazam (16)

118.4 ± 3.4 *** + 114% 7.9 ± 0.9 * + 68%

15 mice per group Student's t-test: NS = not significant; * = p <0.05; *** = p <0.001

Oregano extract 1 to 10 and 60 mg / kg increased the time spent in the lighted compartment, in

15 comparison with vehicle control (+ 28%, and + 35%, p = 0.0506 respectively, close to significance). It tended to increase the number of crosses at 10 mg / kg (+ 51%, p = 0.0888) but had no clear effect at 30 and 60 mg / kg. Thus, it can be assumed that oregano extract 1 has a moderate anxiolytic effect, as measured in the dark-lit cage test in the mouse.

Example 11: Effect of oregano extract 4 on the Porsolt forced swimming test in the mouse after oral feeding by subchronic esophageal probe

This test, which detects antidepressant activity, was performed in the same manner as described in example 7, except that the route of administration of the compound and the doses were different. Oregano extract 4 was evaluated at 3 doses (75, 150 and 300 mg / kg), administered orally (p.o.) 24, 5, and 1 hour before the test, and compared with a vehicle control group. The oregano extract 1 so administered was dissolved in "vegetable" corn oil

25 free of tocopherol). Vehicle (p.o.) was administered to the control group, while imipramine (32 mg / kg), p.o .; dissolved in water) to a separate group as a reference compound, 24, 5 and 1 hour before the test.

The mice were individually placed in cylinders (height = 24.5 cm, diameter = 19.5 cm) containing 13.5 cm of water (22 ° C) from which they could not escape. The mice were left in the water for 6 minutes, automatically examined by a video camera and monitored by an available software system.

30 commercially (VideoMot2, TSE Systems GmbH, Germany), and the duration of immobility was measured during the last 4 minutes.

Ten mice were studied for each of the 5 groups. The data were analyzed by comparison of the treated groups and the positive control group with the vehicle group using the Analysis of Variance (ANOVA) and the Bonferroni post-hoc test.

35 Table 6: Results of the forced swimming test in the mouse after subchronic administration of 3 concentrations of oregano extract 1.

TREATMENT

DURATION OF IMMOBILITY (s)

(mg / kg) p.o .., -24, -5, -1 h

Mean ± s.e.m. % change from control

Vehicle

129.35 ± 11.10 -

Oregano Extract 1 (75) Oregano Extract 1 (150) Oregano Extract 1 (300)

76.74 ± 46.02 87.79 ± 14.08 102.06 ± 13.69 -41% * -32% * -21% NS

Imipramine (32)

64.59 ± 8.26 -fifty % ***

10 mice per group

Student t-test: NS = not significant; * = p <0.05; *** = p <0.001

Thus, oregano extract 4 significantly reduced immobility time, compared to the group of

5 control, in 41% and 32% respectively in the low and intermediate dose groups. Overall, a significant effect of oregano extract 4 was observed. Imipramine (32 mg / kg ip) administered under the same experimental conditions, significantly reduced immobility behavior, compared to vehicle control (-50%, p < 0.001).

These results demonstrate that oregano extract 4 (75 and 150 mg / kg, p.o.) has an efficacy similar to

10 tricyclic antidepressant imipramine, in its ability to significantly reduce behavior related to depression.

Example 12: Effect of oregano extract 4 on serotonin levels in the hippocampus measured by in vivo microdialysis

Male Sprague-Dawley rats (250-320 g) were housed in groups of 4-5 under temperature conditions (21 ± 2ºC) and

15 humidity (55 ± 10%) controlled with free access to food and water (with the lights on from 07.00 to 19.00). The rats were anesthetized using chloral hydrate (400 mg / kg ip) and with a simple microdialysis probe (BASi type MD2200, 2 mm membrane, 30,000 Daltons cut-off point) implanted in the dorsal hippocampus using a stereotactic frame in the coordinates following (face-flow -4.5 mm; medium-lateral -2.5 mm; back-ventral -4.5 mm of the bregma and the surface of the dura according to Paxinos & Watson 6) and fixed

20 in position with dental cement. Body temperature was maintained at 36 ° C using a heating pad and monitored by means of a digital rectal thermometer. The microdialysis probe was perfused with artificial cerebrospinal fluid (aCSF) at 1 μl / min and extracellular monoamine levels were determined by collecting perfused medium samples every 15 min and tested using high performance liquid chromatography (HPLC) With electrochemical detection.

The mobile phase of HPLC (0.5 M EDTA, 0.1 M monochloroacetic acid pH 3.1, 0.15 g / l sodium octyl sulfate, 5% acetonitrile, 0.7% tetrahydrofuran ) was pumped through the system at 70 μl / min. Monoamines were separated using a 1 x 100 mm ODS 3 mm reverse phase capillary column with 5 μl injection loop and detected using an epsilon electrochemical detector (BASi) with a carbon glass electrode set at +650 mV versus a reference electrode Ag / AgCl. Dialysate peaks were identified by comparison of

30 peak elution times with reference standards and were quantified according to the measurement of the peak area using linear regression analysis. The detection limits for 5HT and 5 HIAA were defined as the amount of sample that produced a double peak area of background noise per unit time and was approximately 0.1 fmol / sample in both cases.

Preliminary studies showed that after implantation of the dialysis probe, the 5HT level was

35 initially high due to the release of platelets activated by blood coagulation caused by surgery, but within 150 minutes of completion of surgery the baseline level of 5HT was practically constant. For this reason, injections were routinely administered 150, 210 and 270 min after surgery and perfused samples were collected up to 60 minutes after the final injection.

For routine testing, two rats were prepared, one of which was used to test the oregano extract and the

40 other as control sample (vehicle). Due to the variability of the baseline 5HT release between the assays and the variability in the efficiency of the microdialysis probes to detect 5HT in the extracellular fluid, these data were normalized according to the two values obtained immediately before the first injection ( that is the samples at the moments of 135 and 150 min). Data from replicated studies for each compound are expressed as% of the baseline level, as is normal practice for microdialysis studies. In order to determine the effect of each dose of the extract on the total amount of 5HT released, the area under the curve (AUC) was estimated using the trapezoid method throughout the sampling period subsequent to each dose administration and they compared the values for the test compound / extract with the appropriate control by two-way ANOVA (being the treatment factors and Dose) followed by post-hoc dose comparisons

5 individuals using the Bonferroni t test. All statistical analyzes were performed using Graphpad Prism.

Oregano extract 4 (10, 30 and 60 mg / kg, ip) was dissolved in saline solution containing 0.2% (w / v) hydroxypropylmethylcellulose, while the reference compound, fluoxetine (3, 10 and 30 mg / kg, ip) was dissolved in saline. Two control groups were further investigated, to which saline was administered which

10 contained 0.2% (w / v) hydroxypropyl methylcellulose or saline alone, respectively.

Table 7: Effect of Oregano Extract 1 on cumulative 5HT in each dosing period and expressed in relation to the basal level of 5HT measured immediately before the injection of the first dose of compound. Injections were performed at 150, 210 and 270 min after surgery. The dose of each compound is expressed as mg / kg. Data are expressed as the mean ± s.e.m. (n = 5-6). The statistical analysis for each dose of compound is compared against the corresponding time period for the appropriate vehicle, be saline.

or saline solution + 0.2% hydroxypropylmethylcellulose, using the Bonferroni t test and for each compound globally by two-way ANOVA.

TREATMENT (mg / kg)

Injection time after surgery (min) 5-HT LEVEL (% baseline)

Mean ± s.e.m.

Statistical significance

Saline solution

150 210 270 84 ± 4 71 ± 9 65 ± 7

0.2% (w / v) hydroxypropylmethyl cellulose in saline solution

150 210 270 80 ± 7 68 ± 4 67 ± 5

Oregano Extract 4 (10) Oregano Extract 4 (30) Oregano Extract 4 (60)

150 210 270 88 ± 3 82 ± 5 75 ± 5 † † †

Fluoxetine (3) Fluoxetine (10) Fluoxetine (30)

150 210 270 85 ± 4 131 ± 9 160 ± 7 ††† ††† ††† *** ***

5 - 6 rats per two-way ANOVA group: versus global saline control group; † p <0.05, ††† p <0.001 Bonferroni post hoc test: versus saline control group; *** p <0.001

Example 13: Inhibition of the absorption of dopamine by oregano extract 1

The estimate of the cumulative release of 5HT measured as AUC within 60 min after administration

20 of each treatment dose (Table 7) as analyzed by two-way ANOVA also indicated a significant overall effect of treatment with oregano extract 4 (F (1.30) = 5.61; p <0.05) .

The actions of several neurotransmitters, including dopamine, are regulated by their rapid absorption and clearance of synaptic joints by plasma membrane transport proteins. The dopamine transporter in the central dopaminergic neurons is responsible for the recovery of up to 90% of the released neurotransmitter. Monoamine transporters are high affinity targets for a number of psychoactive agents such as cocaine, amphetamine, and antidepressants. These agents, by blocking the transporters and consequent prevention of neuronal absorption, raise the concentration levels of extracellular neurotransmitters in both the central and peripheral nervous systems, contributing to their

30 behavioral and autonomic effects.

CHO-Ki / hDAT cells, which expressed the human dopamine transporter (hDAT) were plated before the assay. The cells (2 x 105 / ml) were incubated with oregano extract and / or vehicle in modified Tris-HEPES buffer pH 7.1 at 25 ° C for 20 minutes before the addition of 50 nM [3H] dopamine for 10 minutes. The specific signal was determined in the presence of 10 μM nomifensin. The cells were then solubilized with 1% SDS lysis buffer. The reduction in the absorption of [3H] dopamine by 50% or more (≥ 50%) relative to vehicle controls indicates significant inhibitory activity. The pure compounds (nomifensin thimoquinol) were screened at 10 concentrations up to 100 µM: 0.00316, 0.01, 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6 and 100 µM. The oregano extract was screened at 10 concentrations up to 100 μg / ml: 0.00316, 0.01, 0.0316, 0.1, 0.316, 1,

5 3.16, 10, 31.6 and 100 μg / ml. These same concentrations were applied simultaneously to a separate group of untreated cells and were evaluated for possible cytotoxicity induced by the compound only if a significant inhibition of absorption was observed.

Table 8: IC50 values measured for inhibition of dopamine reabsorption in CHO-Ki cells transfected by oregano extract 1, and its volatile thimoquinol component. Data are represented as mean ± s.e.m., where the

IC50 is expressed as μM (or nM) for individual compounds and as μg / ml for oregano extract.

Compound

IC50

* Nomifensin

11 nM

Thimoquinol

65.6 ± 1.2 μM (n = 2) *

Oregano Extract 1

6.3 μg / ml

* Indicates the standard reference agent used.

References:

Giros, et al. 1992 Mol. Pharmacol 42: 383-390 15 Pristupa, et al 1994 Mol. Pharmacol 45: 125-135

Example 14: Inhibition of norepinephrine absorption by oregano extract

The actions of several neurotransmitters, including norepinephrine, are regulated by their rapid absorption and clearance of synaptic joints by plasma membrane transport proteins. The norepinephrine transporter in central adrenergic neurons is responsible for the recovery of up to 90% of released neurotransmitter. Monoamine transporters are high affinity targets for various psychoactive agents such as cocaine, amphetamine, and antidepressants. These agents, by blocking the transporters and consequent prevention of neuronal absorption, raise the concentration levels of extracellular neurotransmitters in both the central and peripheral nervous systems, contributing to their

25 behavioral and autonomic effects.

MDCK dog kidney cells stably expressing the recombinant human norepinephrine transporter were plated one day before the test. The cells (2 x 105 / ml) were pre-incubated with the oregano extract and / or vehicle in modified Tri-HEPES buffer of pH 7.1 at 25 ° C for 20 minutes, after which 25 nM [3H] norepinephrine was added to incubation for 10 minutes. The cells contained in the well were then washed twice, solubilized with 1% SDS lysis buffer and the lysate was counted to determine the absorption of [3 H] norepinephrine. The specific signal was determined in the presence of 10 μM desipramine. The reduction in the absorption of [3H] norepinephrine by 50% or more (≥ 50%) in relation to vehicle controls indicates significant inhibitory activity. The pure compounds (desipramine) were screened at 10 concentrations up to 100 μM: 0.00316, 0.01, 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6 and 100 μM. Oregano extract

35 1 was screened at 10 concentrations up to 100 μg / ml: 0.00316, 0.01, 0.0316, 0.1, 0.316, 1, 3.16, 10, 31.6 and 100 μg / ml. These same concentrations were applied concurrently to a separate group of untreated cells and were evaluated for possible cytotoxicity induced by the compound only if a significant inhibition of absorption was observed.

Table 9: IC50 values measured for inhibition of norepinephrine reabsorption in MDCK cells transfected 40 by oregano extract 1. Data are presented as mean ± sem, where IC50 is expressed as nM for individual compounds and as μg / ml. for oregano extract.

Inhibitor

IC50

* Desipramine

1.9 nM

Oregano extract

13.6 μg / ml

* Indicates the standard reference agent used. Reference:

45 Galli, et al. 1995. J. Exp. Biol. 198: 2197-2212 The concentrations of "free" carvacrol (aglycone) and "total" carvacrol (aglycone + conjugated form) were determined in 64 rat plasma samples. Four male rats and four female rats received a single dose of 800 mg of oregano extract 4 per kg of body weight per oral esophageal tube, respectively. The application solution was prepared in corn oil at a concentration of 200 mg of extract per gram of formulation. Be

5 collected plasma samples at 0, 0.5, 1, 2, 3, 4, 6, 8, 24 hours and 48 hours (terminal) after application by esophageal probe of at least 3 male animals and 3 female animals .

The sample analysis was performed with a liquid chromatography system - tandem mass spectrometry (LC / MS / MS), using a column change system for in-line purification and desalination of the samples.

After the addition of the internal standard (D2-thymol) and the precipitation of the protein (in the case of the "free" analyte) or

10 pre-digestion by β-glucuronidase followed by precipitation of the protein (in the case of the "total" analyte), the samples were injected into a Waters XTerra ™ MS C18 guard column used as a purification column, and then transferred to a Waters XTerra ™ MS C18 column. Detection was performed using tandem mass spectrometry in MRM mode.

Calibration and quality control samples were prepared in 5% bovine albumin serum solution and

15 human plasma. The day-to-day behavior was monitored with the results of the quality control samples (QC) within each analytical lot. The lower limit of quantification was set at 10.0 ng / ml for "free" carvacrol and 20.0 ng / ml for "total" carvacrol.

A kinetic study was performed after oral administration of oregano extract 4 to male and female rats.

20 The objective of this analytical study was the determination of "free" and "total" concentrations of carvacrol in rat plasma samples. A total of 64 samples were analyzed successfully.

Plasma samples were collected at different times after application of the esophageal probe. The dose administered was 800 mg / kg of oregano extract 4 in corn oil.

The "free" carvacrol concentrations measured varied from undetectable to 50,100 ng / ml, and the "total" carvacrol concentrations from undetectable to 50,000 ng / ml.

Table 10: Determination of free and total carvacrol in plasma samples of female ND * rats not detected

ID number

Sample Name (Sex / # Rat / Time) Carvacrol Free (ng / ml) Total Carvacrol (ng / ml)

one

F  one 0 * ND * ND

2

F one 0.5 30.9 2160

3

F one one 192 4220

4

F one 2 64.3 829

5

F one 3 128 3540

6

F one 4 246 4480

7

F one 6 135 4000

8

F one  8 159 3810

9

F one 24 127 5850

10

F one Terminal (48h) * ND <LOQ = 20.0 ng / ml

eleven

F 2 0 * ND * ND

12

F 2 0.5 5120 14800

13

F 2 one 6050 18600

14

F 2 2 840 3550

fifteen

F 2 3 228 1780

16

F 2 4 102 1160

17

F 2 6 91.9 1920

18

F 2 8 1630 6800

19

F 2 24 8.91 1530

twenty

F 2 Terminal (48h) * ND <LOQ = 20.0 ng / ml

twenty-one

F 3 0 * ND * ND

22

F 3 0.5 4090 11000

2. 3

F 3 one 3610 10000

24

F 3 2 1360 6470

25

F 3 3 174 1360

26

F 3 4 50.3 928

27

F 3 6 48.3 1140

28

F 3 8 <LOQ = 10.0 ng / ml 541

29

F 3 24 <LOQ = 10.0 ng / ml 1680

30

F 3 Terminal (48h) * ND <LOQ = 20.0 ng / ml

31

F 4 0 * ND <LOQ = 20.0 ng / ml

32

F 4 0.5 50100 50,000

LOQ * below the lower limit of quantification: LOQ * (free carvacrol) = 10.0 ng / ml LOQ * (total carvacrol) = 20.0 ng / ml

Table 11: Determination of free and total carvacrol in plasma samples of male ND * rats not detected

ID number

Sample Name (Sex / # Rat / Time) Carvacrol Free (ng / ml) Total Carvacrol (ng / ml)

33

M one 0 * ND * ND

3. 4

M one 0.5 1320 18700

35

M one one 1820 25200

36

M one 2 199 5590

37

M t 3 51.5 3010

38

M one 4 30.4 2750

39

M one 6 83.4 6290

40

M one 8 185 8610

41

M one 24 68.1 8460

42

M one Terminal (48h) * ND <LOQ = 20.0 ng / ml

43

M 2 0 * ND * ND

44

M 2 0.5 1530 11100

Four. Five

M 2 one 1790 13500

46

M 2 2 166 1260

47

M 2 3 58.4 665

48

M 2 4 40 1040

49

M 2 6 980 8080

fifty

M 2 8 156 2780

51

M 2 24 <LOQ = 10.0 ng / ml 1820

52

M 2 Terminal (48h) * ND <LOQ = 20.0 ng / ml

53

M 3 0 * ND * ND

54

M 3 0.5 103 2310

55

M 3 one 1150 8550

56

M 3 2 584 6380

57

M 3 3 1580 11200

58

M 3 4 316 2900

59

M 3 6 100 2040

60

M 3 8 140 3630

61

M 3 24 <LOQ = 10.0 ng / ml 2750

62

M 3 Terminal (48h) * ND <LOQ = 20.0 ng / ml

63

M 4 0 * ND * ND

64

M 4 0.5 55.9 4960

Example 16: Preparation of a soft gelatin capsule

LOQ * below the lower limit of quantification: LOQ * (free carvacrol) = 10.0 ng / ml LOQ * (total carvacrol) = 20.0 ng / ml

A soft gelatin capsule comprising the following ingredients can be prepared:

Ingredient

Amount per Capsule

Oregano Extract

500 mg

Lecithin

50 mg

Soy oil

250 mg

Two capsules can be administered per day for 3 months to a human adult for the treatment of mild chronic dysthymia. 10 Example 17: Preparation of a soft gelatin capsule A soft gelatin capsule comprising the following ingredients can be prepared:

Ingredient

Amount per Capsule

Oregano Extract

200 mg

Spring oil at night

300 mg

Vitamin B6

100 mg

One capsule may be taken per day, preferably during the second half of the menstrual cycle, for 14 days for the treatment of premenstrual syndrome and premenstrual dysphoric disorder.

15 Example 18: Preparation of a flavored instant soda

Ingredient

Quantity [g]

Oregano Extract

0.9

Sucrose, fine powder

922.7

Ascorbic acid, fine powder

2.0

Citric acid, anhydrous powder

55.0

Lemon Essence

8.0

Trisodium citrate, anhydrous powder

6.0

Tricalcium phosphate

5.0

β-Carotene 1% CWS of DNP AG, Kaiseraugst, Switzerland

  0.4

Total quantity

1000

All ingredients are mixed and screened through a 500 μm sieve. The resulting powder is placed in an appropriate container and mixed in a tubular mixer for at least 20 minutes. To prepare the drink, 125 g of the mixed powder obtained is mixed with enough water to produce 1 liter of drink.

The ready-to-drink soda contains approx. 30 mg of oregano extract per serving (250 ml). As a booster and for general well-being, 2 services per day (240 ml) are recommended.

Example 19: Preparation of an unbaked reinforced cereal bar

Ingredient

Quantity [g]

Oregano Extract

         0.95

Sugar

114.55

Water

54.0

Salt

1.5

Glucose syrup

130.0

Invert sugar syrup

95.0

Sorbitol syrup

35.0

Palm Almond Fat

60.0

Baking grease

40.0

Lecithin

1.5

Hydrogenated Palm Oil

2.5

Dried and cut apple

63.0

Cornflakes

100.0

Crispy Rice Grains

120.0

 Crunchy Wheat Grains

90.0

Toasted hazelnut

40.0

Skimmed milk powder

45.0

Apple Essence 74863-33

2.0

Citric acid

5.0

Total quantity

1000

The oregano extract is previously mixed with skim milk powder and placed in a planetary bowl mixer. Corn flakes and crunchy grains of rice are added, and mixed gently. The dried and cut apples are then added. In a first cooking pan, sugar, water and salt are mixed in the amounts indicated above (solution 1). In a second cooking pan, glucose syrups, invert sugar and 5 sorbitol are mixed in the amounts indicated above (solution 2). A mixture of baking butter, palm kernel butter, lecithin and emulsifier is the fat phase. Solution 1 is heated to 110 ° C. Solution 2 is heated to 113 ° C and then cooled in a cold water bath. After that, solutions 1 and 2 are combined. The fat phase is melted at 75 ° C in a water bath. The fat phase is added to the combined mixture of solutions 1 and

2. Apple flavor and citric acid are added to the sugar-fat liquid mixture. The liquid mass is added to

10 dry ingredients and mix well in the planetary bowl mixer. The dough is placed on a marble plate and laminated to the desired thickness. The dough is cooled to room temperature and cut into pieces. The unbaked cereal bar contains approx. 25 mg of oregano extract per serving (30 g). For general wellbeing and energy intake, 1-2 cereal bars can be taken per day.

Example 20: Dry dog food comprising oregano extract or its volatile components for stress relief and revitalization of the dog

A commercial basal diet for dogs (eg Mera Dog "Brocken", MERA-Tiernahrung GmbH, Marienstraβe 80-84, D-47625 Kevelaer-Wetten, Germany) is sprayed with an oregano extract solution in an amount sufficient to be administered to a individual a daily dose of 50 mg per kg of body weight, based on the weight of oregano extract or the concentrate of its volatile components. The food composition is dried so that

20 contains approximately 90% by weight dry matter. For an average dog weighing 10 kg of body weight that consumes approx. 200 g of dry feed per day, dog food contains approx. 2500 mg of oregano extract or its volatile components / kg of food. For dogs of greater weight, the feed mixture is prepared analogously.

Example 21: Wet cat food comprising oregano extract or its volatile components

25 A commercial basal diet for cats (eg Happy Cat "Adult", Tierfeinnahrung, Südliche Hauptstraβe 38, D86517 Wehringen, Germany) is mixed with a solution of oregano extract or its volatile components in an amount sufficient to give an individual a dose daily of 100 mg per kg of body weight, based on the weight of dried or concentrated oregano extract of its volatile components. For an average cat of 5 kg of body weight that consumes approx. 400 g of wet food, cat food contains approx. 1250 mg / kg

30 oregano extract. The food composition is dried so that it contains approximately 90% by weight dry matter.

Example 22: Treatments for dogs containing oregano extract

Commercial dog treatments are sprayed (e.g. Mera Dog "Biscuit", MERA-Tiernahrung GmbH, Marienstraβe 80-84, D-47625 Kevelaer-Wetten, Germany) with an oregano extract solution or its

35 volatile components in an amount sufficient to administer to the treatments 5-50 mg per g of treatment, based on the weight of the dried oregano extract or the concentrate of its volatile components. The food composition is dried so that it contains a dry matter of approximately 90% by weight.

Example 23: Treatments for cats containing oregano extract

Commercial cat treatments are sprayed (e.g. Whiskas Dentabits for cats supplied by Whiskas,

40 Master-foods GmbH, Eitzer Str. 215, 27283 Verden / Aller, Germany) with a solution of oregano extract or its volatile components in an amount sufficient to administer to the treatments 5-50 mg per g of treatment, based on the weight of dried oregano extract or the concentrate of its volatile components. The food composition is dried so that it contains a dry matter of approximately 90% by weight.

Claims (7)

1. Use of an orally administrable dietary or pharmaceutical composition comprising an extract obtained from plants of the genus Origanum by extraction with a solvent or supercritical fluids, hydro-distillation to obtain essential oils, or extraction / distillation with hot gases in a process for: 5 maintenance or improvement of attention and concentration, memory and ability to remember, ability to learning, language processing, problem solving and intellectual functioning, short-term memory improvement, increased mental alertness,       increased concentration and mental acuity, 10 intensification of mental surveillance, reduction of mental fatigue, reinforcement of mental health, maintenance of a balanced cognitive function, regulation of hunger and satiety and regulation of motor activity, 15 use as an antidepressant, mood / vitality enhancer, stress reliever, anxiety reducer, stress reducer, sadness reducer, unhappiness / discontent reducer, irritability reducer, dysphoria reducer, behavior reducer obsessive-compulsive, and / or relaxing, or use as a preventive or normalizer of alterations of circadian rhythms, with the proviso that the composition does not include the combination of oregano and Crataegus extracts. Use of a composition according to claim 1, wherein the oregano extract is obtained from Origanum vulgare. 3. Use of a composition according to any of claims 1-2, wherein the composition is selected from the group consisting of dairy products, yogurts, reinforced food, cereal bars, pastries, cakes, cookies, dietary supplements, tablets, pills, granules, dragees, capsules, formulations 25 effervescent, non-alcoholic beverages, soft drinks, sports drinks, fruit juices, lemonades, fundamentally aqueous drinks, teas, milk-based drinks, liquid food, soups, and muesli drinks.

Four.

Use of a composition according to any of claims 1-3 which is a veterinary use.

5.

Use of a composition according to any of claims 1-4, wherein the oregano extract comprises at least 30% by weight of carvacrol.

6. Use of a composition according to any of claims 1-5, wherein the oregano extract comprises thimoquinone in an amount in the range of 0 to 30% by weight. 7. Use of a composition according to any of claims 1-6, wherein the oregano extract comprises at least 50% by weight of carvacrol and 0 to 25% by weight of thimoquinone. 8. Use of a composition according to any of claims 1-7, wherein the oregano extract contains a simple volatile component selected from the group consisting of carvacrol and thimoquinone. 9. Use of a dietary or pharmaceutical composition comprising an extract obtained from plants of the genus Origanum by extraction with a solvent or supercritical fluids, hydro-distillation to obtain essential oils, or extraction / distillation as hot gases in a process for: use as a norepinephrine reuptake inhibitor, dopamine reuptake inhibitor, and / or 40 dual inhibitors of serotonin, norepinephrine and / or dopamine absorption, or use as a triple norepinephrine, serotonin and / or dopamine absorption inhibitor , with the proviso that the composition does not include the combination of oregano and Crataegus extract.